In contrast to human immunodeficiency virus type 1 (HIV-1) V3, the simian immunodeficiency virus (SIV) V3 does not appear to be variable in peripheral blood leukocytes of persistently infected macaques as well as among laboratory isolates. However, considerable genetic variation in V3 in tissues of rhesus macaques that died from AIDS following infection with cloned SIVmac239 has been observed. The pattern of amino acid variation in SIV V3 was not random (similar to those in HIV-1 V3). The amino acids comprising the predicted crown and stem regions of the V3 loop were well conserved; however, the regions flanking the crown on both sides were highly variable. Molecularly cloned SIV containing one of the naturally mutated V3 sequences in gp120 showed higher replicative and cytopathic ability for T-lymphocytes than SIVmac239. The mutated V3 is more positively charged and hydrophilic than V3 of SIVmac239, which are properties similar to V3 of HIV-1 with high replicative and cytopathic ability for T-lymphocytes. These observations strongly suggest that SIV V3 might be subjected to similar selective pressures that occur on HIV-1 V3, and indicate that V3 in SIV plays a role in the pathogenesis of SIV infections, as it does in HIV-1. To determine the biological significance of SIV V3 for viral cell tropism the V3 of T cell-tropic and macrophage-tropic cloned SIV was exchanged with the naturally mutated V3 sequences, and V3 recombinant viruses were generated. In human T cell lines, Ev/T3-mtv3 recombinant virus replicated as well as SIVmac239, in contrast to the inability of Ev/T3 to replicate in human T cell lines. Similarly, 239-mtv3 displayed dramatically reduced replicative potential in human T cell lines compared to SIVmac239. In rhesus macaque lymphocyte cultures, Ev/T3-mtv3 showed remarkably reduced replicative potential compared to Ev/T3, and the 239-mtv3 recombinant replicated better than SIVmac239. These data demonstrate that the V3 sequences of Ev/T3 and SIVmac239 determine the replicative potential of these viruses in human T cell lines and rhesus lymphocytes in vitro.